Experiments are proposed to determine the role of the transmembrane domain (TMD) in membrane fusion. We found that a specific TMD sequence element is required for fusion by the vesicular stomatitis virus fusion protein, named G; specifically, a TMD gly residue is essential for complete fusion, but not for hemifusion (mixing of the outer leaflets of the reacting membrane bilayers). In addition, a survey of the sequences of TMDs of other virus fusion proteins showed that gly residues are found in most of them (24 of 28 surveyed), in contrast to a control group of non-fusion proteins. A general hypothesis is formulated, which proposes that the TMD sequence specifies a fusion-competent TMD conformation, which mediates a late step in fusion. Suitably placed glycine residues are sufficient, but not always necessary, for TMDs to assume this conformation. Experiments are proposed to test and extend this hypothesis. (1) The positional and residue specificity of the required TMD gly residue in VSV G protein will be determined using saturation mutagenesis at the TMD gly residue sites, and by introducing gly residues at different locations in an inactive background. (2) The contribution of the VSV TMD gly residues to helicity and helix stability of the TMD will be assessed by circular dichroism (CD), nuclear magnetic resonance (NMR), and differential scanning calorimetry (DSC) using model peptides that possess the authentic VSV G protein TMD, compared with fusion inactive analogs lacking gly residues. The peptides will be characterized in detergent solution, and in several membrane bilayer environments. (3) In order to test the generality of the gly requirement, the role of the TMD gly residue in the influenza fusion protein, HA, will be determined, using similar approaches. (4) The TMD sequence requirements for fusion will be determined for those viral fusion proteins that lack gly residues. CD, NMR and DSC experiments with model TMD peptides will be carried out, similar to those for VSV. These experiments are expected to lead to a unified and comprehensive understanding of the role of the TMD in both viral and cellular fusion reactions.